Establishing a system for de novo generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) would open a unique opportunity to study human HSC development and provide a novel source of therapeutic cells for blood disease. Achieving this goal requires a detailed understanding of cellular and molecular pathways that lead to blood formation from hPSCs and identification of the immediate precursors of multipotential hematopoietic cells.
Avian, mouse and human embryonic studies demonstrated that definitive HSCs which give rise to all lineages of an adult hematopoietic system are generated in the aorta-gonad-mesonephros (AGM) region and are located at the ventral aspect of the dorsal aorta (de Bruijn et al., 2002; Ivanovs et al., 2011; Pardanaud et al., 1996; Taoudi and Medvinsky, 2007). In this area, hematopoietic cells arise from a unique population of endothelial cells known as hemogenic endothelium (HE) through an endothelial-hematopoietic transition (EHT) (Boisset et al., 2010; Jaffredo et al., 2000; Zovein et al., 2008). Dynamic tracing and imaging studies conducted in vivo demonstrated that EHT represents a continuous process in which cells with endothelial characteristics gradually acquire hematopoietic morphology and phenotype (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010).
Definitive hematopoiesis in the AGM region is preceded by primitive hematopoiesis in the yolk sac, which initially generates primitive erythrocytes, megakaryocytes, and macrophages (Palis et al., 1999; Xu et al., 2001). The second wave of yolk sac hematopoiesis, defined as erythromyeloid hematopoiesis, is associated with expansion of erythroid precursors producing adult β hemoglobin and unilineage and multilineage myeloid precursors (Palis et al., 1999). Although the concept of HE was coined based on observations of blood formation within the aorta, it is also known that endothelium lining nascent capillaries in the yolk sac (Ferkowicz et al., 2003) and possibly vitelline and umbilical arteries (Yokomizo and Dzierzak, 2010) have the capacity to generate blood as well.
The demonstrations of HSC formation from endothelium emphasized the need for access to well-defined populations of HE cells in hPSC cultures in order to develop technologies for de novo generation of HSCs from human induced pluripotent (hiPSCs) or embryonic stem cells (hESCs). In the embryo definitive HE can be identified based on anatomical location, morphology, and expression of Runx1 (Jaffredo et al., 2010; North et al., 1999; North et al., 2002). Because these criteria cannot be entirely applied to cells differentiated in vitro, the precise identification of HE in hPSC cultures remained as a significant challenge. Although VE-cadherin+CD41a− and/or CD45− phenotype is commonly used for detection and isolation of HE, it has very limited utility in human PSC cultures since it covers the entire population of endothelial cells, does not fully exclude hematopoietic cells, and does not discriminate between endothelial lineages with restricted primitive hematopoietic and multipotential definitive potentials. In addition, the direct mesodermal precursor of HE with definitive hematopoietic potential remains largely unknown.
Needed in the art is an improved method of generating HE progenitors and generating novel populations of hemogenic endothelial cells, angiogenic blood cells or endothelial progenitors.